Survival of Environmental Mycobacteria in Acanthamoeba polyphaga

Free-living amoebae in water are hosts to many bacterial species living in such an environment. Such an association enables bacteria to select virulence factors and survive in adverse conditions. Waterborne mycobacteria (WBM) are important sources of community- and hospital-acquired outbreaks of nontuberculosis mycobacterial infections. However, the interactions between WBM and free-living amoebae in water have been demonstrated for only few Mycobacterium spp. We investigated the ability of a number (n = 26) of Mycobacterium spp. to survive in the trophozoites and cysts of Acanthamoeba polyphaga. All the species tested entered the trophozoites of A. polyphaga and survived at this location over a period of 5 days. Moreover, all Mycobacterium spp. survived inside cysts for a period of 15 days. Intracellular Mycobacterium spp. within amoeba cysts survived when exposed to free chlorine (15 mg/liter) for 24 h. These data document the interactions between free-living amoebae and the majority of waterborne Mycobacterium spp. Further studies are required to examine the effects of various germicidal agents on the survival of WBM in an aquatic environment.     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.     

Mycobacterium avium Bacilli Grow Saprozoically in Coculture with Acanthamoeba polyphaga and Survive within Cyst Walls

Protozoans are gaining recognition as environmental hosts for a variety of waterborne pathogens. We compared the growth of Mycobacterium avium, a human pathogen associated with domestic water supplies, in coculture with the free-living amoeba Acanthamoeba polyphaga with the growth of M. avium when it was separated from amoebae by a 0.1-μm-pore-size polycarbonate membrane (in a parachamber). Although viable mycobacteria were observed within amoebal vacuoles, there was no significant difference between bacterial growth in coculture and bacterial growth in the parachamber. This suggests that M. avium is able to grow saprozoically on products secreted by the amoebae. In contrast, Legionella pneumophila, a well-studied intracellular parasite of amoebae, multiplied only in coculture. A comparison of amoebae infected with L. pneumophila and amoebae infected with M. avium by electron microscopy demonstrated that there were striking differences in the locations of the bacteria within amoebal cysts. While L. pneumophila resided within the cysts, M. avium was found within the outer walls of the double-walled cysts of A. polyphaga. These locations may provide a reservoir for the bacteria when environmental conditions become unfavorable.     

Defined minimal growth medium for Acanthamoeba polyphaga

Nutritional requirements of Acanthamoeba polyphaga (strain PD) were compared to those reported for A. castellanii. Although A. polyphaga and A. castellanii have essentially the same minimal amino acid requirements--arginine, methionine, leucine, isoleucine, and valine--A. polyphaga cannot utilize acetate as sole carbon source, but A. castellanii can if the medium is supplemented with glycine.     

Defined Minimal Growth Medium for Acanthamoeba polyphaga

Nutritional requirements of Acanthamoeba polyphaga (strain PD) were compared to those reported for A. castellanii. Although A. polyphaga and A. castellanii have essentially the same minimal amino acid requirements–arginine, methionine, leucine, isoleucine, and valine–A. polyphaga cannot utilize acetate as sole carbon source, but A. castellanii can if the medium is supplemented with glycine.     

Survival of environmental mycobacteria in Acanthamoeba polyphaga

Free-living amoebae in water are hosts to many bacterial species living in such an environment. Such an association enables bacteria to select virulence factors and survive in adverse conditions. Waterborne mycobacteria (WBM) are important sources of community- and hospital-acquired outbreaks of nontuberculosis mycobacterial infections. However, the interactions between WBM and free-living amoebae in water have been demonstrated for only few Mycobacterium spp. We investigated the ability of a number (n = 26) of Mycobacterium spp. to survive in the trophozoites and cysts of Acanthamoeba polyphaga. All the species tested entered the trophozoites of A. polyphaga and survived at this location over a period of 5 days. Moreover, all Mycobacterium spp. survived inside cysts for a period of 15 days. Intracellular Mycobacterium spp. within amoeba cysts survived when exposed to free chlorine (15 mg/liter) for 24 h. These data document the interactions between free-living amoebae and the majority of waterborne Mycobacterium spp. Further studies are required to examine the effects of various germicidal agents on the survival of WBM in an aquatic environment.     

Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.     

Replication and Long-Term Persistence of Bovine and Human Strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: Physiological Status and Susceptibility to Chemical Inactivation

[This corrects the article on p. 2422 in vol. 58.].     

Interactions of some common pathogenic bacteria with Acanthamoeba polyphaga

Protozoan grazing is a major trophic pathway whereby the biomass re-enters the food web. Nonetheless, not all bacteria are digested by protozoa and the number known to evade digestion, resulting in their environmental augmentation, is increasing. We investigated the interactions of Bacillus cereus, Enterococcus faecalis, Enteropathogenic Escherichia coli (EPEC), Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and methicillin-sensitive Staphylococcus aureus (MSSA), with the amoeba, Acanthamoeba polyphaga. There was evidence of predation of all bacterial species except L. monocytogenes and S. aureus, where extracellular numbers were significantly higher when cultured with amoebae compared with growth in the absence of amoebae. Intracellular growth kinetic experiments and fluorescent confocal microscopy suggest that S. aureus survived and may even multiply within A. polyphaga, whereas there was no apparent intra-amoebal replication of L. monocytogenes and higher numbers were likely sustained on metabolic waste products released during coculture.     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Yield Constant for Acanthamoeba polyphaga in Batch and Continuous Culture1

ABSTRACT. A two-stage chemostat modified to accommodate the growth of adhesive organisms was used to determine the yield constant, Y, of a representative soil amoeba, Acanthamoeba polyphaga, utilizing as its prey Pseudomonas paucimobilis. The first stage consisted of a glucose-limited bacterial culture in steady state. The second stage consisted of a simplified predator-prey system, nongrowing bacteria serving as the limiting substrate for amoebae. A refined methodology to more accurately determine Y was developed, and Y for Acanthamoeba polyphaga in batch and continuous culture was determined to be 19.1%.     

Bacteriolytic activities of the free-living soil amoebae,Acanthamoeba castellanii,Acanthamoeba polyphaga andHartmannella vermiformis

Bacteriolytic activities of axenically grown free-living soil amoebaeAcanthamoeba castellanii, Acanthamoeba polyphaga andHartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined. A spectrophotometric assay revealed that the specific bacteriolytic activities of bothAcanthamoeba species were higher as those of the threeHartmannella strains.Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus andPseudomonas fluorescens were more easily lysed than the other bacteria tested.Agrobacterium tumefaciens, Klebsiella aerogenes andSerratia marcescens were hardly affected at all by the amoebal bacteriolytic activities. Among the Gram-negative bacteria we observed differences in lysis sensitivity while the Gram-positive bacteria tested were sensitive to lysis. Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3–10 was performed to separate the bacteriolytic isoenzymes of amoebae. Bacteriolytic patterns were shown by using an activity assay in which lysis bands were formed in the agar/bacteria gel-overlay. The activity assay revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae. Distinct differences between typical pI points of bacteriolytic activities inAcanthamoeba andHartmannella were shown. Bacteriolytic activities ofHartmannella were more pronounced and observed in the isoelectric points (pI) range of 4.0–9.3 while forAcanthamoeba the range was pI 4.5–8.9.Abbreviations IEF isoelectric focusing - PAA-IEF polyacrylamide-isoelectric focusing - CCAP culture collection of algae and protozoa - AS amoeba saline medium - pI isoelectric points     

Nitrogen Mineralization by Acanthamoeba polyphaga in Grazed Pseudomonas paucimobilis Populations

Nitrogen mineralization was studied in a simple grazing system in which the protozoan Acanthamoeba polyphaga was grown with the bacterium Pseudomonas paucimobilis (two soil organisms isolated from the shortgrass prairie in northern Colorado). In different experiments, either carbon or nitrogen was adjusted to be in limiting amounts. When carbon was limiting, grazers were almost entirely responsible for nitrogen mineralization, with bacteria themselves contributing little. When nitrogen was limiting, nitrogen mineralization by grazers permitted continued growth by the grazed bacteria and a greater bacterial biomass production. The increased growth of the grazed bacteria did not result in an increased total amount of carbon used, but the grazed bacteria used carbon more efficiently than the ungrazed bacteria.     

Protozoan Acanthamoeba polyphaga as a potential reservoir for Campylobacter jejuni

We showed by a laboratory experiment that four different Campylobacter jejuni strains are able to infect the protozoan Acanthamoeba polyphaga. C. jejuni cells survived for longer periods when cocultured with amoebae than when grown in culture alone. The infecting C. jejuni cells aggregated in amoebic vacuoles, in which they were seen to be actively moving. Furthermore, a resuscitation of bacterial cultures that were previously negative in culturability tests was observed after reinoculation into fresh amoeba cultures. After spontaneous rupture of the amoebae, C. jejuni could be detected by microscopy and culturability tests. Our results indicate that amoebae may serve as a nonvertebrate reservoir for C. jejuni in the environment.     

Protozoan Acanthamoeba polyphaga as a Potential Reservoir for Campylobacter jejuni

We showed by a laboratory experiment that four different Campylobacter jejuni strains are able to infect the protozoan Acanthamoeba polyphaga. C. jejuni cells survived for longer periods when cocultured with amoebae than when grown in culture alone. The infecting C. jejuni cells aggregated in amoebic vacuoles, in which they were seen to be actively moving. Furthermore, a resuscitation of bacterial cultures that were previously negative in culturability tests was observed after reinoculation into fresh amoeba cultures. After spontaneous rupture of the amoebae, C. jejuni could be detected by microscopy and culturability tests. Our results indicate that amoebae may serve as a nonvertebrate reservoir for C. jejuni in the environment.     

Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus

Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the “stargate”, allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane–containing viruses.     

Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure

The association between Legionella pneumophila and the free-living amoeba Acanthamoeba polyphaga was studied. Intracellular growth of L. pneumophila within amoebic trophozoite was confirmed by kinetic growth experiments, light and electron microscopy. Cysts produced from infected trophozoites were found to protect the legionellas from at least 50 mg/l free chlorine. The ability of L. pneumophila to survive within the cysts of A. polyphaga is suggested as a possible mechanism by which the organism evades disinfection and spreads to colonize new environments.